The genome sequence of the Bordered Straw, Heliothis peltigera (Denis & Schiffermüller) 1775

We present a genome assembly from an individual male Heliothis peltigera (the Bordered Straw; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 332.8 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.3 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,114 protein coding genes.


Background
The Bordered Straw Heliothis peltigera is a Noctuid moth, but although it has the characteristic thick body and wingshape seen in this family, it is brighter coloured than most of its dull brown cousins, with a ground colour ranging from strawyellow, early in the season, to darker orange-brown, as increasing temperatures in the larval stages causes the adults to become darker (Waring et al., 2017).It is usually more distinctly marked than similar species with a bold grey kidney mark and diagnostic dark diffuse blotches on the leading edge of the forewing near the kidney and adjacent to the darker band on the outer edge: the similar Scarce Bordered Straw or Old World Bollworm Helicoverpa armigera lacks both of these diagnostic markings, and Eastern Bordered Straw Heliothis nubigera has only a smaller more diffuse marking near the kidney, with a paler ground colour and three or more distinct small dots near the outer edge (Skinner & Wilson, 2009;Waring et al., 2017).
Within the United Kingdom Heliothis peltigera is considered to be an immigrant species (Waring et al., 2017), but it is common in tropical and subtropical areas of Africa, where it is a major agricultural pest and, with climate change, is spreading north into Europe (Ragionieri et al., 2017).The larvae cause damage to crops such as safflower Carthamus tinctorius, tobacco Nicotiana tabacum, grapevine Vitus spp., cotton Gossypium spp., stone and citrus fruit trees, fodder crops, medicinal and ornamental plants (Dunkelblum & Kehat, 1992).Consequently, the synthesis and production of pheromones by H. peltigera (Altstein, 2004;Ragionieri et al., 2017) and the moths' response to them (Dunkelblum & Kehat, 1992) have been the subject of intense research.More recently molecular methods have been used to identify a strain of Helicoverpa armigera single nucleopolyhedrovirus (HearNPV-TR), which infects H. peltigera, and is similar to other viruses which have been used for the biological control of other insect pests (Eroglu et al., 2020).
We present a chromosomally complete genome sequence for Heliothis peltigera, based on one specimen collected using a mercury vapour light trap in a rural garden in the hamlet of Bratton, near Minehead, in Somerset, as part of the Darwin Tree of Life Project.

Genome sequence report
The genome was sequenced from a male Heliothis peltigera (Figure 1) collected from Bratton, Somerset, UK (51.20,.A total of 75-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 4 missing joins or mis-joins, increasing the scaffold number by 3.23%. The final assembly has a total length of 332.8 Mb in 31 sequence scaffolds with a scaffold N50 of 11.8 Mb (Table 1).The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.99%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).The Z chromosome was identified based on synteny with Craniophora ligustri (GCA_905163465.1)(Boyes et al., 2023).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition and nucleic acid extraction
A male Heliothis peltigera (specimen ID Ox002232, ToLID ilHelPelt1) was collected from Bratton, Somerset, UK (latitude 51.20, longitude -3.51) on 2022-06-20, using a light trap.The specimen was collected by Denise Wawman (University of Oxford) and identified by Liam Crowley (University of Oxford), and then snap-frozen on dry ice.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences SEQUEL II instrument.Hi-C data were also generated from head tissue of ilHelPelt1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.(Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

MITOS
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).This work was done using Nextflow (Di Tommaso et al  et al., 2015) were calculated.
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Heliothis peltigera assembly (GCA_958496145.1) in Ensembl Rapid Release at the EBI.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material • Legality of collection, transfer and use (national and international) Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer 1.In the last paragraph of Background, the authors claim that they present a "chromosomally complete genome sequence" for Heliothis peltigera.Given that there are still some gaps inside the chromosomes, and that the presence of telomeres on chromosomal ends has not been ascertained, I would suggest avoiding emphasizing the genome sequence is complete.Instead, "near-complete" is a more cautious statement.
2. The authors state that "The Z chromosome was identified based on synteny with Craniophora ligustri(GCA_905163465.1) (Boyes et al., 2023)."Given that sex chromosome transitions are quite common in some animal lineages, could the authors explain a little further in the manuscript why this is workable?For example, is that because the sex chromosomes are highly conserved across Noctuidae species?This information is not clear to the readers.
3. All the software tools used for building and assessing the genome assembly have been provided in methods and table 3.But to allow replication by others, the parameters of each tool are required as well.Even if default parameters are used, it is worthy to mention somewhere in the manuscript.I suggest that the authors may provide the comments of parameters used for each tool in table 3, especially when some tools are run with default settings, but some are not.
4. The protein-coding genes are annotated for the genome assembly, which are valuable for future studies.However, the completeness of the gene set is not assessed.At least, presenting the assessment for the gene set using BUSCO will be informative.
Next Generation Sequence techniques.A total of 332.8 megabases in span were assembled through the genome assemble.Totally 17,114 protein-coding genes and 17,323 number of gene transcripts were observed through the annotation.Authors have utilized appropriate techniques for DNA isolation, library construction for sequencing and used proper software for assembly and annotation.

Comment 1:
The author's name of the species with year can be included in the first sentence of the background."The Bordered Straw Heliothis peltigera (Denis & Schiffermüller) 1775 is a Noctuid moth…" There is confusion in this sentence "but although it has the characteristic thick body and wingshapeseen in this family".Please delete the sentence or can be modified.

Comments 2:
Authors have used the word kidney instead of reniform in the background of the species.Actually, it's a kidney shaped or renal shaped (reniform) marking not kidney.So, the second sentence of the background can be changed as "It is usually more distinctly marked than similar species with a bold grey reniform mark and diagnostic dark diffuse blotches on the leading edge of the forewing near the reniform and adjacent to the darker band on the outer edge: the similar Scarce Bordered Straw or Old World Bollworm Helicoverpa armigera lacks both of these diagnostic markings, and Eastern Bordered Straw Heliothis nubigera has only a smaller more diffuse marking near the reniform, with a paler ground colour and three or more distinct small dots near the outer edge (Skinner & Wilson, 2009;Waringet al., 2017)".

Comment 3:
Authors have given the genus name Heliothis peltigera full form throughout the article.For the first time the genus name can be given in full form then subsequently can be given in short form like H. peltigera

Comment 4:
The second paragraph first sentence fourth line "remove the comma (,) after and…..

Comment 5:
Total length of the mitochondrial genome sequence must be provided in the text.
Above all, I confirm that the manuscript meets the necessary scientific standard and is suitable for indexing" Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Molecular biology
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Genome assembly of Heliothis peltigera, ilHelPelt1.1:metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 332,843,978 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (15,076,996 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (11,813,164 and 8,357,070 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the lepidoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilHelPelt1_1/dataset/ilHelPelt1_1/snail.

Figure 3 .
Figure 3. Genome assembly of Heliothis peltigera, ilHelPelt1.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilHelPelt1_1/dataset/ilHelPelt1_1/blob.

Figure 4 .
Figure 4. Genome assembly of Heliothis peltigera, ilHelPelt1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilHelPelt1_1/dataset/ilHelPelt1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Heliothis peltigera, ilHelPelt1.1:Hi-C contact map of the ilHelPelt1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=YWF6i8BXRhmu_mJj5vyPDg.

Table 1 . Genome data for Heliothis peltigera, ilHelPelt1.1. Project accession data
Assembly metric benchmarks are adapted from column VGP-2020 of "Table 1: Proposed standards and metrics for defining genome assembly quality" from Rhie et al. (2021).
(Jay et al., 2023)high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.In sample preparation, the ilHelPelt1 sample was weighed and dissected on dry ice(Jay et al., 2023).Tissue from the thorax was homogenised using a PowerMasher II tissue disruptor(Denton  et al., 2023a).HMW DNA was extracted using the Automated MagAttract v2 protocol(Oatley et al., 2023a).DNA was sheared into an average fragment size of 12-20 kb in a